Biotechnology and Molecular Biology
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| Genetics is considered what kind of science? | basic science
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| Genetic engineering is what kind of science? | applied science
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| DNA unwinding | DNA unwinds itself at just below boiling point; exposes nucleotides making DNA easier to ID, replicate, and transcribe. Renatures as it cools (into double stranded form)
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| Restriction endonucleases | clips polynucleotide strands crosswise; originate in bacterial cells; Recognize foreign DNA & can break phosphodiester bonds b/t adjacent nucleotides on both strands; protects against incompatible DNA of bacteriophages/plasmids
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| Restriction endonucleases | Used in labs to cleave the strand at desired sites
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| Palindromes | sequences of DNA that are identical when read from the 5’ to 3’ direction of one strand and the 5’ to 3’ direction on the other strand. (ex. eye, racecar)
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| Endonuclease naming | named by combining first letter of bacterial genus, first two letters of the species, and endonuclease number. EcoRI is the first endonuclease in E. coli and HindIII is the third nuclease in Haemophilus influenzae
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| Sticky ends | the short tails of severed DNA
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| Restriction fragments | pieces of DNA produced by restriction endonucleases
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| restriction fragment length polymorphisms (RFLPS) | restriction fragments of differing lengths; allow direct comparison of the DNA of two different organisms at a specific site
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| Ligase | seals sticky ends together by rejoining the phosphate-sugar bonds cut by endonucleases
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| Reverse transcriptase | enzyme; converts RNA into DNA
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| Complementary DNA (cDNA) | can be made from mRNA, tRNA, rRNA, et al. Provides valuable means of synthesizing eukaryotic genes from mRNA transcripts.
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| Gel electrophoresis | produces readable pattern of DNA fragments; sample placed in soft agar gel, subjected to electric current; DNA phosphate grps give molecule neg charge, causes DNA to move toward pos pole in gel; Rate of movement depends on size of fragments (large = slow)
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| Hybridize | uniting of two different nucleic acids at their complementary regions; different combos possible (single DNA with other single RNA or DNA, RNA with other RNA; allows for specially formulated oligonucleotide tracers (gene probes)
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| Gene probes | oligonucleotide tracers; short stretch of DNA of a known sequence that will base-pair w/ stretch of DNA w/ complementary sequence; detects specific sequences in unknown samples; Reactions revealed by placing photographic film in contact w/ test reaction.
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| Southern blot method | technique that separates fragments of DNA using electrophoresis and identifies them by hybridization
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| FISH | fluorescent in situ hybridization; probes applied to intact cells and observed microscopically for the presence and location of specific genetic marker sequences on genes; effective way to locate genes on chromosomes
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| Methods used to size, synthesize, and sequence DNA | DNA sequencing, sanger method, polymerase chain reaction (PCR) DNA sequencing
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| Sanger method | based on synthesis and analysis of complementary strand of DNA in a test tube.
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| Polymerase chain reaction | Xerox machine for DNA; increases amt DNA in sample w/out cultures or carrying out complex purification techniques; “plucks” a DNA “needle out of a haystack;” same events as DNA synthesis (unzipping, addition of primers, action of DNA polymerase)
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| Primers | synthetic oligonucleotides; landmarks for where DNA amplification should begin
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| DNA polymerases | processing must be carried out at a high temperature to keep strands separated, so polymerases are isolated from thermophilic bacteria
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| Steps in PCR cycle | it is through cyclic repetition of these steps that DNA becomes amplified; denaturation, priming, extension
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| Denaturation | DNAs formed in first cycle become amplicons upon denaturization; heating target DNA to 94 degrees C to separate it into two strands; then system is cooled to between 50 and 65 depending on nucleotide sequence
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| Priming | primers added in a concentration that favors binding to the complementary strand of test DNA; reaction prepares the two DNA strands now called amplicons for synthesis.
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| Extension | DNA polymerase and raw materials in the form of nucelotides are added
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| Recombinant DNA technology | to deliberately remove genetic material from one organism and combine it with that of a different organism.
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| Cloning | removal of selected gene from genetic donor followed by its propagation in different host organism; Gene inserted into vector (usually plasmid/virus) that will insert the DNA into cloning host
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| Clones | genetic clones; have same parental DNA
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| Vectors | receiver of propagated gene from another organism; usually a plasmid or virus; it will insert the DNA into the cloning host.
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| Cloning hosts | usually a bacterium or yeast that can replicate the gene and translate it into the protein product for which it codes.
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| Technical aspects of recombinant DNA and gene cloning | finding target gene on donor chromosome and isolating it. DNA removed from cells, separated into fragments; fragments inserted into vector and cloned; cloned frags undergo southern blotting and are probed to ID desired sequences (long process). OR gene c
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| Characteristics of cloning vectors | must be capable of carrying a significant piece of donor DNA; must be readily accepted by the cloning host, must have a promoter in the front of the cloned gene.
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| Charon phage | modified phage vector; missing large sections of its genome, so it can carry a large segment of foreign DNA
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| Biochemical Products of Recombinant DNA Technology | Human growth hormone, Insulin, Clotting factor VIII
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| Genetically Modified Organisms | Recombinant organisms produced through introduction of foreign genes are called transgenic or genetically modified organisms (GMOs).
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| Recombinant microbes | Modified bacteria and viruses
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| Gene therapy | correct/repair a faulty gene in humans suffering from a fatal or debilitating disease.
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| Ex vivo | normal gene is cloned in vectors (retroviruses or adenoviruses). Tissues from patient are incubated w/ these viruses to transfect them with the normal gene. The transfected cells are then reintroduced into the patient’s body by transfusion.
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| In vivo | therapy skips the intermediate step of incubation; instead, naked DNA or a virus vector is directly introduced into the patient’s tissues.
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| DNA technology as genetic medicine | treatment prevents transcription or translation of a gene that is unwanted.
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| Antisense DNA | Targeting mRNA; when binds to its particular mRNA the double-stranded RNA is inaccessible to the ribosome, resulting in loss of translation of that mRNA; the reading of that mRNA transcript on ribosomes will be blocked and the gene product will not be syn
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| DNA fingerprinting | A unique picture of a genome
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| What is a restriction enzyme? | clips polynucleotide strands crosswise at selected positions; can recognize/clip palindromes, recognize foreign DNA; can break phosphodiester bonds; originate in bacterial cells; used in labs to cleave the strand at desired sites
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| What evolutionary advantage does a restriction enzyme confer on the bacterium that makes it? | Protection from foreign DNA (such as that from phages).
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| What is a clone? | A clone is an animal that is genetically the same as its parent. It shows no recombination and no variation.
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| Briefly describe the Sanger method of sequencing DNA | DNA to be sequenced cut into large number of short fragments and separated; test strands denatured to expose single strands to act as templates for complementary strands; fragments divided into 4 separate tubes w/ fluorescent/radioactive primers indicatin
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| Describe DNA fingerprinting | The exact way nucleotides are combined is unique for each organism; DNA fingerprinting emphasizes these differences and arrays the genome in a pattern for comparison. It can be used for medical diagnosis, genetic ancestry information, and forensics.
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| What is a limitation of DNA fingerprinting? | The sample DNA to be tested could be contaminated or degraded by the time it undergoes testing.
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| When DNA is used as a probe what rules define what the probe will bind to? What factors affect this binding? | The DNA will pair with a complementary sequence; Base-pair rules (AT, GC)
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| What is a STR? | Short Tandem Repeats found in noncoding portions of genome; they do not have protein functionality.
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| How are STRs used in criminal investigations? Dose STR analysis have the same limitations as DNA fingerprinting? | STRs are used in genetic fingerprinting to create a genetic profile of a person for comparison, usually based on DNA collected from a crime scene or victim. It has the same limitations as DNA fingerprinting.
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| What is a recombinant? | A recombinant organism is produced through the introduction of foreign genes that have been inserted into their genome purposefully. They could also be called transgenic or genetically modified.
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| Describe a useful medical product made by recombinant DNA technology. | Recombinant human insulin; it was previously obtained from pigs.
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| When was the last time you cloned something? What was it? Why was it a clone? | Working with my lab unknown;created a streak plate of the organism from a broth sample. It is a clone because the colonies on the streak plate are genetically identical to the parent cells that I obtained from the broth sample.
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